Islet cell cluster produced from human umbilical cord mesenchymal stem cells

a mesenchymal stem cell and islet cell technology, applied in the field of cell culture methods, can solve the problems of inability to obtain enough mass purified iccs, inability to produce iccs, degeneration of iccs, etc., to achieve the effect of promoting or maintaining the survival and function of target cells, preventing type i diabetes, and maintaining the survival and function of iccs

Active Publication Date: 2015-04-28
NAT TAIWAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0007]The present invention provides a cell culture method, particularly to a co-culture method for human mesenchymal stem cells and target animal cells, in order to solve the problem that animal cells are not easy to survive alone upon culturing. The activity and function of target animal cells can be maintained. The present invention also provides a method for using a stem cell conditioned medium to culture animal cells. Finally, the present invention also provides a method to induce the transformation of human islet-like cell clusters from human stem cells and application thereof.
[0030]The embryonic stem cells need a special culture environment to growth ex vivo. For example, they need leukemia inhibitor factor (LIF) to promote the proliferation of stem cells and inhibit the differentiation of stem cells. Because the human umbilical cord mensenchymal stem cells (HUMSCs) do not need special environment to growth ex vivo, they are easier to be cultured compared to the embryonic stem cells. Therefore, the present invention employs HUMSCs to co-culture with other target cells, in order to maintain the survival and function of co-cultured target cells. When HUMSCs are used for co-culture, the cell clusters can be protected from damage. Consequently, it can be used as a novel cell culture. Before the transplantation or experiment of target cell clusters, it can be used to increase the number of target cells, and maintain the survival and function of target cells.
[0031]HUMSCs are isolated from the human umbilical cord, which can be easily obtained and processed compared to embryonic and bone marrow stem cells, and can be cultured without the utilization of special hormone. HUMSCs possess excellent self-renewing, repair and proliferative capabilities. As for the culture of HUMSCs, the category and concentration of secreted hormone are easy to be controlled and verified, so it is easy to carry on the assessment of scientific experiment. In addition, the safety is very high, so long as the mother and infant are healthy, there will be no infection problem for HUMSCs, which can be used for mass production, and it is not necessary to check whether it is infected by virus again.
[0032]According to the cell culture method of the present invention, the co-culture of HUMSCs can maintain the survival and function of target cells. As exampled by the culture of rat islet-like cell clusters, when the rat ICCs are co-cultured with HUMSCs, the number of rat ICCs and secretion amount of insulin can be increased for 3 months continuously. However, the rat ICCs which do not co-culture with HUMSCs will be damaged gradually and died within 12 days.
[0034]In addition, according to the cell culture method of the present invention, when the stem cell conditioned medium is used to culture target cells, the effect is equivalent to the result of using HUMSCs for co-culture. The active ingredients in stem cell conditioned medium (SCM) is similar to the active ingredients in co-culture medium, including the interleukin-6 (IL-6), tissue inhibitor of metalloproteinase-1 (TIMP-1), tissue inhibitor of metalloproteinase-2 (TIMP-2), monocyte chemoattractant protein-1 (MCP-1), growth related oncogene (GRO), hepatocyte growth factor (HGF), insulin-like growth factor binding protein 4 (IGFBP-4) and interleukin-8 (IL-8). For example, if the stem cell conditioned medium is used to transform stem cells into ICCs, it can maintain the survival and function of ICCs for more than 2 weeks, and still possesses the insulin secretion capability. It can be said that the culture by the stem cell medium has the same effect with respect to the co-culture of HUMSCs. The transformed ICCs still possesses the insulin secretion capability, so they can be transplanted to the liver of a subject in need thereof, and applied to inhibit type I diabetes further.

Problems solved by technology

But because the supply of ICCs transplantation is insufficient, the ICCs are lost during separation and purification process, it is difficult to get enough mass purified ICCs, which is often one of the reasons to fail for the fail of ICCs transplantation.
In addition, ex vivo culture of ICCs is very difficult, and the common culture method cannot exceed 3 to 5 days.
Because in the present common ex vivo culture method, ICCs will be denatured, dead, and unable to be utilized

Method used

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  • Islet cell cluster produced from human umbilical cord mesenchymal stem cells
  • Islet cell cluster produced from human umbilical cord mesenchymal stem cells
  • Islet cell cluster produced from human umbilical cord mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

embodiment 1

of HUMSCs

[0043]With the consent of the parents, fresh human umbilical cords were obtained after birth and collected in HBSS buffer solution (Gibco) at 4° C. HUMSCs were obtained as previously described by Hu et al. (Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopaminergic neurons in vitro: Potential therapeutic application for parkinsonism. Stem Cells 24:115-124; 2006). Briefly, the mesenchymal tissue of Wharton's jelly of the umbilical cord was diced into cubes of about 0.5 cm3 and centrifuged at 250×g for 5 minutes; and then the mesenchymal tissue was treated with collagenase type I (Sigma) at 37° C. for 18 hours, washed and further digested with 2.5% trypsin (Gibco) at 37° C. for 30 minutes. The digested mixture was then passed through a 100 μm filter to obtain cell suspensions. Then the cell suspensions were centrifuged at 250×g for 5 minutes. The HUMSCs culture medium consisted of Dulbecco's modified Eagle's medium / F12 (DMEM / F12, 25 mM glucos...

embodiment 2

of Co-Culture System

[0044]Seven-day postnatal Sprague-Dawley rats were used for preparation of pancreatic cells and ICCs formation. The study was approved by The Animal Research Committee of The College of Medicine, National Taiwan University. After rats were sacrificed, pancreases were removed and cut into small pieces, and then incubated in 2 ml medium supplemented with 3 mg collagenase for 20 min at 37° C. Re-suspended to the collagenase-free medium and centrifuged at 1500 r.p.m. for 3 min. The final pellets were suspended in 12 ml DMEM / F12, 2% fetal bovine serum, 1 mM glutamine, and 10 mM nicotinamide. For preparation of co-culture system, HUMSCs were cultured in 6-well plates with 80% confluence at 37° C. Place the inserts into each prepared well with the membrane towards the well bottom, ensuring no air is trapped. Add 2 ml of single pancreatic cell suspensions to the interior of each insert (pore size 3.0 μm or 0.2 μm, Nunc).

[0045]During pancreatic cells culture, less than 30...

embodiment 3

tification Assay

[0047]To test whether ICCs co-cultured in embodiment 2 still have functional characteristics of islets, we will inspect whether the activity of insulin secreted by cell is normal. The supernatants of ICCs culture and ICCs / HUMSCs co-culture medium were harvested every 3 days for insulin detection using a rat insulin-ELISA-kit (enzyme linked immunosorbent assay kit, Mercodia, Sweden). After acquiring the medium, washed plate 5 times with PBS and changed medium every 3 days.

[0048]See FIG. 2, on day 6, the insulin levels of ICCs culture and ICCs / HUMSCs co-culture are 89.43±3.32 and 89.97±2.12 μg / L, respectively. However, the insulin levels in ICCs culture medium rapidly declined on day 9 (4.23±0.64 μg / L) and day 12 (2.87±0.09 μg / L). Meanwhile, the insulin levels in ICCs / HUMSCs co-culture still gradually increased on day 9 (92.35±2.18 μg / L), day 12 (145.40±26.85 μg / L), and even lasted to day 90 (160.30±31.92 μg / L). In addition, there was no human insulin detected in ICCs / ...

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Abstract

The invention relates to a cell culture method, particularly to a co-culture method for human mesenchymal stem cells and target animal cells, in order to solve the problem that animal cells are not easy to survive alone upon culturing. The invention also provides a method for using a stem cell conditioned medium to culture animal cells. The invention also provides a method to induce the transformation of human fetal islet-like cell clusters from human stem cells and its application thereof.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]The invention relates to a cell culture method, particularly to a co-culture method for human mesenchymal stem cells and target animal cells.[0003]2. Description of the Prior Art[0004]Type I diabetes (also called insulin relied type diabetes) is mainly caused by the abnormality of pancreatic islet cell which is responsible for secreting the insulin. Though it only accounts for 5-10% of all cases of diabetes, the patients become dependent on daily injection of insulin in order to control the illness condition for life, which will influence the life of patients seriously. So, there are a lot of researches on type I diabetes.[0005]It is generally acknowledged that islet-like cell clusters (ICCs) can generate insulin. Transplantation of islet-like cell clusters is the best method to cure type I diabetes at present. But because the supply of ICCs transplantation is insufficient, the ICCs are lost during separation and purifi...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): A61K48/00A61K38/27
CPCA61K38/27
Inventor LIU, SHING-HWACHAO, KUO-CHINGCHAO, KUO-FANG
Owner NAT TAIWAN UNIV
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